This proposal describes the development of rapid assays for detection and optimization of low molecular weight gp41 inhibitors effective against Human Immunodeficiency Virus (HIV-1) fusion. Fusion inhibitors possess excellent characteristics for interrupting HIV transmission and preventing disease progression, since they block initial infection of healthy cells and cell-to-cell spread of infection. Peptide fusion inhibitors are highly potent, due to the large surface area formed by their interaction with a hydrophobic groove in the coiled coil domain of gp41. Yet they possess some undesirable characteristics as drugs, such as high cost and susceptibility to proteolysis. There are currently no highly active small molecule inhibitors of fusion. Most small molecules have been directed towards a hydrophobic pocket on the surface of the coiled coil. We describe screening assays that specifically expose the long coiled coil as a target for small molecules, with the goal of identifying compounds that bind to different sections of the coil. Such molecules could then be linked to form more potent inhibitors, rivaling peptides. To facilitate this approach, we will also develop a structural assay for determining the orientation of bound ligands, necessary information for optimal linking. The primary HTS assay is a mix and measure assay involving modified peptides derived from the N- and C- terminal domain of gp41. The nanomolar association between the peptides is followed by fluorescence intensity readout. Compounds from a library are assessed for activity by their ability to competitively inhibit the peptide association, with a concomitant fluorescence increase. A positive result indicates that a compound is capable of fusion inhibition. The intensity of the fluorescence signal is directly correlated to the compound's potency, over a range of inhibition constants of 0.01-10 5M. Sub-milligram to milligram quantities of each of the peptides is sufficient to screen 300,000 compounds. The secondary assays include evaluation of fusion inhibitory activity of hits in cell - cell and virus - cell assays, and the development of probe peptides in fluorescence or NMR experiments to identify the ligand binding sites. An NMR structure determination method is described that will provide exact details of ligand pose. There are currently no experimental data describing the details of small-molecule - gp41 complexes, and the assays proposed will enable structure-based optimization of hits that has previously relied solely on computational prediction of binding pose. The NMR experiments will be fast, relying on 1D measurement to enable rapid characterization of multiple hits. PUBLIC HEALTH RELEVANCE: The prevalence of HIV infection remains a significant public health problem due to the development of viral strains resistant to current treatments, and due to spread of the virus from individuals who are unaware they are infected. This proposal involves the development of assays capable of screening large chemical libraries for compounds that can inhibit HIV fusion, and characterizing their activity, binding affinity and structure. Fusion inhibitors would be useful in microbicides to prevent infection, or could be given orally to treat infection as an alternative to the currently used fusion inhibitor Enfuvirtide(R), which must be given intravenously.